Gap junctions create direct cell-cell communication in most cell types. These membrane specializations contain one plasma membrane from two apposing cells and tens to thousands of dodecameric connexin channels spanning the two membranes. These form discrete and recognizable cellular structures during quiescent (non-mitotic) phases. Cells dynamically modulate gap junctional communication by regulating the synthesis, transport, gating and turnover of these connexin channels. Current textbook descriptions depicting gap junctions as static, stand-alone structures now are replaced with a new paradigm of connexins, hemichannels, channels and gap junctions as very mobile, dynamic and interactive assemblies. Protein phosphorylation is an important regulatory mechanism by which proteins can control cellular function and/or localization in a process newly termed spatial cell biology. The gap junction protein, connexin43, has a highly regulated life cycle during which several, hierarchical phosphorylation events occur at several specific serine residues in its C-terminus. Different phosphorylation events occur during all stages of the cell cycle and can change which proteins interact with connexin43, the kinetics and/or localization of connexin43 trafficking, assembly, gating, and turnover in a cell cycle stage specific manner that affects important biological processes such as cell migration and proliferation. This project is focused on imaging the elegant interplay between connexin43 phosphorylation, its cellular localization and the cell cycle. The three specific aims of this proposed research are: (1) determine whether certain kinases form complexes with connexin43 at particular stage(s) of its life cycle; (2) to correlate the phosphorylation of specific serine residues with their cellular location singly and in tandem; and (3) to elucidate how specific phosphorylation events are linked to cellular localization during the cell cycle. This proposal focuses on the identification and characterization of connexin trafficking structures using live cell imaging, correlative light and electron tomography with protein tags or probes to produce 3D reconstructions of selectively labeled connexins in cells. Methods for developing and applying multiple probes for correlated light and electron microscopy are essential to the success in imaging trafficking intermediates. In combination with biochemical and inhibitor analyses of wild type and mutant Cx43 proteins, the overall goal is to study these phospho-forms at electron tomographic resolution (~40-60 E) in 3D to determine their morphologies and locations within the context of other cellular components. From these studies, we will gain a mechanistic understanding through advanced imaging how connexin phosphorylation in controls gap junction communication-dependent functions in quiescent cells and during the cell cycle. The importance of this research is driven by the fact that changes in connexin localization and gap junctional communication are part the exquisite control of cellular proliferation, migration and with a loss of growth control during carcinogenesis.